CN1057608C - Affinity chromatographic material, the prepn. method and application thereof - Google Patents
Affinity chromatographic material, the prepn. method and application thereof Download PDFInfo
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- CN1057608C CN1057608C CN95114253A CN95114253A CN1057608C CN 1057608 C CN1057608 C CN 1057608C CN 95114253 A CN95114253 A CN 95114253A CN 95114253 A CN95114253 A CN 95114253A CN 1057608 C CN1057608 C CN 1057608C
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- inhibitor
- arrowhead
- affinity chromatographic
- chromatographic material
- kallikrein
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Abstract
The present invention relates to an affinity chromatography material which satisfies the following structure requirements: R-Arrowhead inhibitors as specific carriers are made of water-insoluble substrate materials, such as various agaroses, and the substrate materials are across linked with the Arrowhead inhibitors to prepare an affinity chromatography material. Accordingly, the affinity chromatography material is used for removing protein enzyme and kallikrein from protein.
Description
The present invention relates to biochemical field.
The arrowhead inhibitor is two kinds of multi-functional crystalline protein enzyme inhibitor A extracting during fresh thatch eats and the potpourri of B, slightly different on these two kinds of protease inhibiting activities, for example inhibitor A is stronger to the white enzyme compatibility of pancreas than suppressing B by force to chymotrypsin and kallikrein compatibility.As seen, the what use is made of thatch eats inhibitor and removes proteinase and kallikrein in the protein better, is the problem that present this field still need solve.
The object of the present invention is to provide a kind of thatch to eat the preparation method of this chromatographic material of affinity chromatographic material of inhibitor one preparing carriers, and adopt this layer folding material to remove the method for proteinase, kallikrein in the protein.
The present invention satisfies following structural requirement:
R_ arrowhead inhibitor
Here R represents a kind of water-fast host material, comprises various types of fine jade sugar.
Being prepared as follows of affinitive material among the present invention:
1, thatch eats the preparation of inhibitor:
Get fresh arrowhead stem tuber, remove crust and clean, shred, add a little deionized water homogenate, filter with 200 order filter clothes, residue stirs with small amount of deionized water, soak filter and and filtrate, adjusting PH to 3.8～4.0.Discard precipitation, get that to reset and add ammonium sulfate to saturation degree be 50%, protein precipitation, matter are abandoned supernatant, precipitation is dissolved in water with the deionized water desalination of dialysing, and thermal denaturation is 5 minutes in rearmounted 65～75 ℃ of water-baths that finish, centrifugal removal precipitation, supernatant is transferred PH to 7.0, with the dialysis of 0.015M phosphate buffer solution.
Get DEAE-cellulose (DEAE-cellulose) gel dress bolt, earlier with 0.015mol/L (PH7.8) phosphate buffer solution balance, above-mentioned solution is added from the post upper end, with 0.05mol/L (PH7.8) phosphate buffer solution flushing post bed, use 0.012mol/L (PH7.8) phosphate buffer solution wash-out then, collect the protein inhibitor eluting peak, eluting peak is no longer increased centrifugal collecting precipitation with the saturating ammonium dialysis of saturated ammonium sulfate to precipitation.Be dissolved in water, with the deionized water desalination of dialysing, after the freeze drying promptly.
2, arrowhead inhibitor and carrier are crosslinked:
Any suitable water-fast host material all can be used as carrier material, for example: Ago-Gel (comprising: agarose 2B, sepharose 4B, agarose 6B and diethylamino agarose), carrier is activated with cyanogen bromide or other activators in advance, add an amount of arrowhead inhibitor, under 1～30 ℃ of condition, react, in the buffer solution that is fit to, place more than 12 hours.The arrowhead inhibitor can be fully and carrier material crosslinked.
The application of affinity chromatographic material of the present invention:
The arrowhead inhibitor is the potpourri of two kinds of multi-functional crystalline protein enzyme inhibitor A and B, these two kinds of protease inhibitors adopt this affinity chromatographic material to can be used for removing contained proteinase and kallikrein in the deproteinize under the PH condition that is fit to suppressing slightly different on the activity.
The agarose 2B (Sephrose 2B, Pharmacia company product) that gets about 1000ml settling volume becomes half-dried thing with the sintered filter funnel suction filtration, with the washing of 0.5mol/L sodium chloride solution, is washed with distilled water to neutrality again.In the ratio of the agarose 2B and the 2g cyanogen bromide of every 10ml settling volume, take by weighing in the mortar of cyanogen bromide and mill, be dissolved in the solution of 0.1mol/L bicarbonate.
With mixed stirring the in the ice bath of sepharose 4B and cyanogen bromide solution, and drip the 2mol/L sodium hydroxide solution, pH is remained between 11.0～11.5, move to room temperature after 10 minutes and placed 10 minutes.To mix liquid and pour Buchner funnel into, and use 0.1mol/L sodium bicarbonate (pH9.5) buffer solution to take out immediately and wash, in 2～3 minutes, wash the buffer solution that is equivalent to 10～15 times of volumes of sepharose 4B, stop activation.
The arrowhead inhibitor of pressing the gel crosslinkable 2.25mg of every 1ml activation calculates, and the arrowhead inhibitor aqueous solution that adds modulated pH to 9.5 slowly stirs under 4 ℃ of conditions placed 12 hours in 2 hours at least.With the crosslinked sepharose 4B of 0.1mol/L sodium bicarbonate (pH9.5) buffer solution washing of about 20 times of volumes, the unconjugated albumen of flush away.With 0.1mol/L hydrochloric acid-glycine buffer solution (pH2.4) flush away non-specific adsorption albumen.Use again on 0.2mol/L glycine buffer solution (pH8.5) the sealing sepharose 4B and protein bound reactive group, putting 4 ℃ slowly stirs and used 0.1mol/L trishydroxymethylaminomethane-hydrochloric acid solution (pH7.5) to take out again in 20 hours to wash, promptly make arrowhead inhibitor-agarose cross-linked composite.Adding 20% ethanol, to put 4 ℃ of preservations standby.
Embodiment II:
Repeat example I, (Sephrose 4B, Pharmacia company product replace agarose 2B to operate equally, make arrowhead inhibitor-sepharose 4B cross-linked composite with sepharose 4B.
Embodiment III:
Repeat example I, replace agarose 2B to operate equally, make arrowhead inhibitor-agarose 6B cross-linked composite with agarose 6B (Sephrose 6B, Pharmacia company product).
Embodiment IV:
Repeat example I, replace agarose 2B to operate equally, make arrowhead inhibitor one diethylamino ethyl agarose cross-linked composite with diethylamino ethyl agarose (DEAE-Sephrose, Pharmacia company product).
Embodiment V:
1. column chromatography:
Get each 1000ml of affinity chromatographic material that adopts above embodiment method preparation, the dress post.Leading to the effluent electricity as equilibrium liquid flushing post bed with the phosphate buffer solution (sodium chloride that contains 0.2mol/L) of 0.1mol/L (pH8.0) in advance leads consistent with the equilibrium liquid electricity.Other gets urokinase (than vigor: 50000IU/mg) make the solution that concentration is 100000IU/ml, regulate pH to 8.0.Each sample 1000ml that goes up, after last sample finished, with the equilibrium liquid flushing post bed of 1000ml, proteinase and kallikrein were adsorbed by affinity column, and urokinase then flows out with solution.Chromatographic column can use the regeneration of 0.1mol/L (pH4.0) sodium acetate buffer solution to reuse.
2. the mensuration of proteinase and kallikrein:
(1) assay method of kallikrein: adopt chromophoric substrate S-2266 determination method [going into Jiang Zhangzi etc., clinical pharmacology (Japan) 29,419, (1981)].
Get 0.2mol/L (pH8.0) trishydroxymethylaminomethane buffer solution 500ul, 37 ℃ are incubated 5～10 minutes, adding inspection product solution 400ul again, mix 37 ℃ of insulations and add the dissolved in distilled water that substrate solution 100ul[25mgS-2266 (Japanese first chemical pharmacy Co., Ltd. product) adds 28.8ml after 2～5 minutes) mixing is incubated 30 minutes for 37 ℃ and adding 50% acetum cessation reaction.The trishydroxymethylaminomethane buffer solution (pH8.0) that other gets the 0.2mol/L that contains Aprotinin 10000～50000IU/L of 500ul replaces the trishydroxymethylaminomethane buffer solution (pH8.0) of 0.2mol/L to operate equally, measure absorbance log (A) as blank at the long 405nm of filter place, be calculated as follows the content of kallikrein:
The content of kallikrein (mU/ml)=A * 9.55
(2) assay method of proteinase: casein decomposes the vitality test method.
Get 37 ℃ of insulations of 1% casein solution mixing that 1.0ml inspection product solution adds 1.0ml after 1 hour, the trichloroacetic acid mixing of adding 50% precipitates unhydrolysed protein, and centrifugal 5 minutes of 3000rpm discards precipitation.Other gets 1.0ml and replaces above-mentioned inspection product solution to operate equally through 100 ℃ of inspection product solution that boiled 2～3 minutes, measures the vigor (O.D.280/ml) of absorbance log (B) as proteinase at the 280nm place as blank.
(3) measure urokinase content (before the processing: kallikrein 2.1mU/ml, proteinase-10 .243O.D.280/ml) of kallikrein and proteinase in the solution before and after the affinity chromatography as stated above, measurement result sees Table one:
Survival rate (%) after the table one affinity column sample preparation
Kallikrein protein enzyme kallikrein protein enzyme
(mU/ml) (O.D.280/ml) M-Spherose 2B 0.0292 0.040 1.4 16.5M-Spherose 4B 0.0181 0.044 0.86 18.1M-Spherose 6B 0.0240 0.035 1.1 14.4M-DEAE-Spherose 0.0070 0.009 0.33 3.7M: represent the arrowhead inhibitor
Embodiment VI:
Repeat embodiment V, replace urokinase to make the solution that concentration is 50000U/ml with urinary trypsin inhibitor (than vigor: 2500U/mg, containing KLK10 .5mU/ml, proteinase-10 .2300.D.280/ml), same operation, measurement result sees Table two:
Survival rate (%) after the table two affinity column sample preparation
Kallikrein protein enzyme kallikrein protein enzyme
(mU/ml) (O.D.280/ml) M-Spherose 2B 0.0227 0.045 0.22 19.8M-Spherose 4B 0.0137 0.026 0.13 11.3M-Spherose 6B 0.0121 0.032 0.12 13.9M-DEAE-Spherose 0.0068 0.014 0.06 6.1M: represent the arrowhead inhibitor
Claims (3)
1, a kind of affinity chromatographic material is characterized in that it satisfies following structural requirement
R_ arrowhead inhibitor
R represents a kind of water-fast host material, comprises various types of fine jade sugar.
2, a kind of preparation method of affinity chromatographic material, it is characterized in that at first water-fast host material R being activated with cyanogen bromide or other activators, add an amount of arrowhead inhibitor, under 1～30 ℃ of condition, react, in the buffer solution that is fit to, place more than 12 hours, thatch aunt inhibitor can be fully and carrier R crosslinked.
3, a kind of application of affinity chromatographic material is characterised in that affinity chromatographic material contained proteinase and the kallikrein in the PH condition that is fit to down isolating protein with claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN95114253A CN1057608C (en) | 1995-12-04 | 1995-12-04 | Affinity chromatographic material, the prepn. method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN95114253A CN1057608C (en) | 1995-12-04 | 1995-12-04 | Affinity chromatographic material, the prepn. method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1141212A CN1141212A (en) | 1997-01-29 |
| CN1057608C true CN1057608C (en) | 2000-10-18 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN95114253A Expired - Lifetime CN1057608C (en) | 1995-12-04 | 1995-12-04 | Affinity chromatographic material, the prepn. method and application thereof |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007081851A2 (en) * | 2006-01-06 | 2007-07-19 | Millipore Corporation | Affinity chromatography matrices and methods of making and using the same |
| WO2010111576A2 (en) * | 2009-03-27 | 2010-09-30 | Hercules Incorporated | Aminated polymers and their use in water-borne compositions |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0203049A1 (en) * | 1985-05-23 | 1986-11-26 | Pharmacia Ab | Method of cross-linking a porous polysaccharide gel |
| EP0295073A2 (en) * | 1987-06-08 | 1988-12-14 | Chromatochem, Inc. | Chromatographic material |
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1995
- 1995-12-04 CN CN95114253A patent/CN1057608C/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0203049A1 (en) * | 1985-05-23 | 1986-11-26 | Pharmacia Ab | Method of cross-linking a porous polysaccharide gel |
| EP0295073A2 (en) * | 1987-06-08 | 1988-12-14 | Chromatochem, Inc. | Chromatographic material |
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| Publication number | Publication date |
|---|---|
| CN1141212A (en) | 1997-01-29 |
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